Although vaccines are widely used and protect against a surprisingly broad spectrum of infectious diseases, protective or therapeutic immunity still cannot be raised against a number of latent and chronic pathogens including Mycobacterium tuberculosis, human immunodeficiency virus, hepatitis C virus, and the Plasmodium protists causing malaria. Conventional approaches that mainly elicit antibody responses have not been successful in providing protective or therapeutic immunity. It is believed that this is due to the fact that epitopes are variable, frequently masked or protected by microbial decoys, or because the dynamic of the infectious cycle may seclude the pathogen into compartments not accessible to antibodies.
Compared to vaccination with inactivated virions or purified subunits, live vaccines induce a broad response that also involves the cellular compartment of the immune system. For safe limitation of a natural infection the vaccine strains are attenuated, but for certain viruses there is a risk of reversion to pathogenic strains (Zurbriggen et al. 2008 in Appl Environ Microbiol 74, 5608-5614) or potential residual virulence for some vaccinees or their contact persons (Marris 2007 in Nat Med 13, 517). In addition, compared to the smallpox eradication program of the 1970s, any pathogenic potential of a vaccine vector may be amplified by the increases in international travel and numbers of immunocompromized individuals. Thus, a greater degree of safety is highly desirable in any novel live vectors (Parrino and Graham 2006 in J Allergy Clin Immunol 118, 1320-1326).
Modern vectored vaccines (Excler et al. 2010 in Biologicals 38, 511-521; Plotkin 2009 in Clin Vaccine Immunol 16, 1709-1719) combine the advantages of live vaccines with the strong safety profile inherent to the highly attenuated vectors, and thus may provide novel therapeutic or protective approaches. Promising vectors are replication deficient alphavirus vectors and highly attenuated poxviruses including modified vaccinia Ankara (MVA), fowlpox (such as strain FP9), and canarypox (ALVAC). These vectors do not replicate in human cells and can therefore be safely given even to immunocompromised recipients (for example (Cebere et al. 2006 in Vaccine 24, 417-425; Dorrell et al. 2007 in Vaccine 25, 3277-3283; Jin et al. 2002 in J Virol 76, 2206-2216; Webster et al. 2005 in Proc Natl Acad Sci USA 102, 4836-4841). They can accommodate large inserts and provide a strong stimulation of the immune system against the vectored antigen (for example (Drillien et al. 2004 in J Gen Virol 85, 2167-2175; Liu et al. 2008 in BMC Immunol 9, 15; Ryan et al. 2007 in Vaccine 25, 3380-3390; Sutter and Moss 1992 in Proc Natl Acad Sci USA 89, 10847-10851; Sutter et al. 1994 in Vaccine 12, 1032-1040).
Disadvantages are directly related to the beneficial properties: the high degree of attenuation necessitates very high numbers of infectious units per dose for full efficacy, and because host range is restricted production requires improved cellular substrates (in some cases from avian donors) or special packaging cell lines.
To illustrate the extent of the industrial challenge with precise numbers and MVA as an example (without limiting this application to MVA only):
Dose requirement is estimated at 108 infectious units of MVA per vaccination (Coulibaly et al. 2005 in Virology 341, 91-101; Gilbert et al. 2006 in Vaccine 24, 4554-4561). For global programs against complex infectious diseases such as HIV or tuberculosis hundreds of million of doses of the highly attenuated poxviruses may be required annually. For comparison, lesser attenuated strains also produced in avian cells include vaccines against measles, mumps and yellow fever; these require only 103, 2×104 and 5.5×104 infectious units per dose, respectively (information from the package inserts of YF-VAX from Sanofi Pasteur and M-M-R II from Merck). The protective dose of the vaccinia strain Dryvax in routine vaccination against smallpox is 2.5×105 pfu (Rotz et al. 2001 in MMWR Recomm Rep 50, 1-25; quiz CE21-27), 400 fold lower than the dose recommended for MVA.
However, production of MVA depends on avian cells. Currently, vaccine strains adapted to avian hosts are produced only in embryonated chicken eggs or on fibroblasts prepared from such eggs, a venerable technology but also associated with certain disadvantages. Because primary cells suffer senescence within few passages they have to be supplied continuously. Differences in timing and preparation may lead to lot variations (Monto et al. 1981 in J Clin Microbiol 13, 233-235; White and Fazekas De St Groth 1959 in J Hyg (Lond) 57, 123-133). The embryonated eggs as source for the fibroblasts are from expensive SPF (specific pathogen free) flocks. The SPF status requires elaborate husbandry, and transport of material across country borders complicates logistics and also cause shortages. Even with SPF precautions in place, contamination with extraneous agents cannot always be prevented. Because time from collection of the embryonated eggs to production of the vaccine is short, testing for extraneous agents is performed on the final bulk (Philipp and Kolla 2010 in Biologicals 38, 350-351). Occasionally, complete vaccine lots have to be discarded when contamination is confirmed by quality testing (Enserink 2004 in Science 306, 385).
Finally, with primary cells it is also not possible to stably express transgenes that may further enhance production of highly attenuated viruses or allow packaging of replication-deficient vectors.
The present inventors have immortalized primary cells from a muscovy duck embryo to replace primary cells as substrate (Jordan et al. 2009 in Vaccine 27, 748-756) and have developed a chemically defined production process for viral vaccines based on this cell line. They have also generated packaging cells based on this cell line (WO 2009/156155). With this technology available one can now expand the development towards vaccine production from a continuous culture in a chemically defined medium and infected with a modern viral vector.
The main challenge at this point is to meet health regulatory guidelines. One of the guidelines that applies here is the World Health Organization Technical Report Series 878 from the year 1998. It is suggested (see the section starting on page 26) that a level of 10 ng of DNA would be acceptable per dose of live vector.
To the knowledge of the present inventors, there are presently no approved live vaccines produced from continuous cell lines. Currently approved live vaccines are produced on primary cells such as chicken embryo fibroblasts (attenuated measles, mumps, yellow fever and influenza viruses), MRC-5 or WI-38 human diploid cell preparations (rubella and varicella viruses), and Vero cells (vaccinia virus and rotavirus). For these cell lines, regulatory procedures with respect to host cell derived components are less stringent, mainly due to the large body of experience that exist. However, as discussed above for chicken embryo fibroblasts, any primary cell preparation has considerable disadvantages and limits in supply. This also applies to Vero cells that may be considered a continuous cell line. However, this line is acceptable for vaccine production only at low cell passage levels (Manohar et al. 2008 in Biologicals 36, 65-72).
Modern continuous cell lines overcome many disadvantages of primary cells but introduce a new challenge, the requirement to define and then to minimize the risk that may be associated with host cell components carried over into the vaccine preparation. Accordingly, the present inventors provide methods of production and purification of live vaccines, which overcome problems of prior art vaccines and provide further advantages.